Plants and seeds of corn variety LH351

ABSTRACT

An inbred corn variety, designated LH351, is disclosed. The invention relates to the seeds of inbred corn variety LH351, to the plants of inbred corn variety LH351 and to methods for producing a corn plant, either inbred or hybrid, by crossing the inbred variety LH351 with itself or another corn variety. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn varieties derived from the inbred LH351.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a new and distinctive corninbred variety, designated LH351. There are numerous steps in thedevelopment of any novel, desirable plant germplasm. Plant breedingbegins with the analysis and definition of problems and weaknesses ofthe current germplasm, the establishment of program goals, and thedefinition of specific breeding objectives. The next step is selectionof germplasm that possess the traits to meet the program goals. The goalis to combine in a single variety or hybrid an improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude higher yield, resistance to diseases and insects, better stalksand roots, tolerance to drought and heat, and better agronomic quality.

[0002] Choice of breeding or selection methods depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc.). For highly heritable traits, a choice ofsuperior individual plants evaluated at a single location will beeffective, whereas for traits with low heritability, selection should bebased on mean values obtained from replicated evaluations of families ofrelated plants. Popular selection methods commonly include pedigreeselection, modified pedigree selection, mass selection, and recurrentselection.

[0003] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding is used to transfer one or a few favorablegenes for a highly heritable trait into a desirable cultivar. Thisapproach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

[0004] Each breeding program should include a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goal and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

[0005] Promising advanced breeding lines are thoroughly tested andcompared to appropriate standards in environments representative of thecommercial target area(s) for three years at least. The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

[0006] These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

[0007] A most difficult task is the identification of individuals thatare genetically superior, because for most traits the true genotypicvalue is masked by other confounding plant traits or environmentalfactors. One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

[0008] The goal of plant breeding is to develop new, unique and superiorcorn inbred varieties and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing and mutations. The breeder has no direct control atthe cellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same corn traits.

[0009] Each year, the plant breeder selects the germplasm to advance tothe next generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop a superior new corn inbred variety.

[0010] The development of commercial corn hybrids requires thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop inbred lines from breedingpopulations. Breeding programs combine desirable traits from two or moreinbred lines or various broad-based sources into breeding pools fromwhich inbred lines are developed by selfing and selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

[0011] Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

[0012] Mass and recurrent selections can be used to improve populationsof either self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

[0013] Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

[0014] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

[0015] Proper testing should detect any major faults and establish thelevel of superiority or improvement over current cultivars. In additionto showing superior performance, there must be a demand for a newcultivar that is compatible with industry standards or which creates anew market. The introduction of a new cultivar will incur additionalcosts to the seed producer, the grower, processor and consumer; forspecial advertising and marketing, altered seed and commercialproduction practices, and new product utilization. The testing precedingrelease of a new cultivar should take into consideration research anddevelopment costs as well as technical superiority of the finalcultivar. For seed-propagated cultivars, it must be feasible to produceseed easily and economically.

[0016] Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F2). Consequently, seed from hybridvarieties is not used for planting stock.

[0017] Hybrid corn seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

[0018] The laborious, and occasionally unreliable, detasseling processcan be avoided by using cytoplasmic male-sterile (CMS) inbreds. Plantsof a CMS inbred are male sterile as a result of factors resulting fromthe cytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

[0019] There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., U.S. Pat. No. 5,432,068 havedeveloped a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility, silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

[0020] There are many other methods of conferring genetic male sterilityin the art, each with its own benefits and drawbacks. These methods usea variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an anti-sense system in which a gene critical to fertilityis identified and an antisense to that gene is inserted in the plant(see, Fabinjanski, et al. EPO 89/3010153.8 publication no. 329, 308 andPCT application PCT/CA90/00037 published as WO 90/08828).

[0021] Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application and genotype specificallyoften limit the usefulness of the approach.

[0022] Corn is an important and valuable field crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding corn hybridsthat are agronomically sound. The reasons for this goal are obviously tomaximize the amount of grain produced on the land used and to supplyfood for both animals and humans. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

[0023] According to the invention, there is provided a novel inbred cornvariety, designated LH351. This invention thus relates to the seeds ofinbred corn variety LH351, to the plants of inbred corn variety LH351and to methods for producing a corn plant produced by crossing theinbred variety LH351 with itself or another corn variety, and to methodsfor producing a corn plant containing in its genetic material one ormore transgenes and to the transgenic corn plants produced by thatmethod. This invention also relates to methods for producing otherinbred corn varieties derived from inbred corn variety LH351 and to theinbred corn varieties derived by the use of those methods. Thisinvention further relates to hybrid corn seeds and plants produced bycrossing the inbred variety LH351 with another corn variety.

[0024] The inbred corn plant of the invention may further comprise, orhave, a cytoplasmic factor that is capable of conferring male sterility.Parts of the corn plant of the present invention are also provided, suchas e.g., pollen obtained from an inbred plant and an ovule of the inbredplant.

[0025] In another aspect, the present invention provides regenerablecells for use in tissue culture or inbred corn plant LH351. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing inbredcorn plant, and of regenerating plants having substantially the samegenotype as the foregoing inbred corn plant. Preferably, the regenerablecells in such tissue cultures will be embryos, protoplasts, meristematiccells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, the presentinvention provides corn plants regenerated from the tissue cultures ofthe invention.

Definitions

[0026] In the description and tables which follow, a number of terms areused. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

[0027] Predicted RM. This trait for a hybrid, predicted relativematurity (RM), is based on the harvest moisture of the grain. Therelative maturity rating is based on a known set of checks and utilizesconventional maturity systems such as the Minnesota Relative MaturityRating System.

[0028] MN RM. This represents the Minnesota Relative Maturity Rating (MNRM) for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

[0029] Yield (Bushels/Acre). The yield in bushels/acre is the actualyield of the grain at harvest adjusted to 15.5% moisture.

[0030] Moisture. The moisture is the actual percentage moisture of thegrain at harvest.

[0031] GDU Silk. The GDU silk (=heat unit silk) is the number of growingdegree units (GDU) or heat units required for an inbred variety orhybrid to reach silk emergence from the time of planting. Growing degreeunits are calculated by the Barger Method, where the heat units for a24-hour period are:$\frac{{GDU} = {\left( {{Max}.{+ {Mini}}} \right) - 50.}}{2}$

[0032] The highest maximum used is 86° F. and the lowest minimum used is50° F. For each hybrid, it takes a certain number of GDUs to reachvarious stages of plant development. GDUs are a way of measuring plantmaturity.

[0033] Stalk Lodging. This is the percentage of plants that stalk lodge,i.e., stalk breakage, as measured by either natural lodging or pushingthe stalks determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

[0034] Root Lodging. The root lodging is the percentage of plants thatroot lodge; i.e., those that lean from the vertical axis at anapproximate 30° angle or greater would be counted as root lodged.

[0035] Plant Height. This is a measure of the height of the hybrid fromthe ground to the tip of the tassel, and is measured in centimeters.

[0036] Ear Height. The ear height is a measure from the ground to theear node attachment, and is measured in centimeters.

[0037] Dropped Ears. This is a measure of the number of dropped ears perplot, and represents the percentage of plants that dropped an ear priorto harvest.

[0038] Allele. The allele is any of one or more alternative forms of agene, all of which alleles relate to one trait or characteristic. In adiploid cell or organism, the two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes.

[0039] Backcrossing. Backcrossing is a process in which a breederrepeatedly crosses hybrid progeny back to one of the parents, forexample, a first generation hybrid F₁ with one of the parental genotypesof the F₁ hybrid.

[0040] Quantitative Trait Loci (QTL). Quantitative trait loci (QTL)refer to genetic loci that control to some degree numericallyrepresentable traits that are usually continuously distributed.

[0041] Regeneration. Regeneration refers to the development of a plantfrom tissue culture.

[0042] Single Gene Converted. Single gene converted or conversion plantrefers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

[0043] Corn variety LH351 is an inbred variety that produces hybridsplants with superior characteristics. Criteria for selections made invarious generations during the development of the variety includedyield, stalk quality, root quality, disease tolerance, late plantgreenness, late plant intactness, ear retention, pollen sheddingability, silking ability and corn borer tolerance. The development ofthe variety can be summarized as follows:

[0044] The development of LH351 was initiated by making the single crossLH211 x LH185. This F1 single cross was then backcrossed with LH185.This F2 combination, LH185² x LH199 was then selfed and the pedigreesystem of plant breeding was then used in the development of LH351.Yield, stalk quality, root quality, disease tolerance, late plantgreenness, late plant intactness, ear retention, pollen sheddingability, silking ability and corn borer tolerance were criteria used todetermine the rows from which ears were selected in the development ofLH351.

[0045] LH211 and LH185, the progenitors of LH351, are both proprietaryfield corn inbred lines developed by Holden's Foundation Seeds, LLC. InDecember 1989, an application was made for plant variety protection ofLH211. On May 31, 1991 LH211 was awarded certificate #9000051. A utilitypatent also protects LH211 (U.S. Pat. No. 5,387,743, issued Feb. 7,1995). In 1993, an application was made for plant variety protection ofLH185. On Feb. 28, 1995 LH1185 was awarded certificate #9400036. Autility patent also protects LH185 (U.S. Pat. No. 5,416,261, issued Mar.16, 1995).

[0046] LH351 has shown uniformity and stability for all traitsdescribed. It has been self-pollinated and ear-rowed a sufficient numberof generations, with careful attention to uniformity of plant type toensure homozygosity and phenotypic stability. The line has beenincreased both by hand (Iowa 2000 and 2001) and sibbed in isolatedproduction fields (Hawaii 2002 and Iowa 2002) with continuedobservations for uniformity and stability. The originating plant breederhas observed LH351 all three generations it has been increased. The lineis uniform, stable and no variant traits have been observed or areanticipated in LH351. An analysis of the traits of inbred corn varietyLH351 is presented below.

[0047] Corn Variety LH351 Description Information

[0048] TYPE: Dent

[0049] REGION WHERE DEVELOPED: Northcentral U.S.

[0050] MATURITY: Days Heat Units From emergence to 50% of plants insilk: 79 1483 From emergence to 50% of plants in pollen 82 1559${{Heat}\quad {Units}}:=\frac{\begin{matrix}\left\lbrack {{{Max}.\quad {Temp}.\left( {\leq {86{^\circ}\quad {F.}}} \right)} +} \right. \\{{{{{Min}.\quad {Temp}}..}\left( {\geq {50{^\circ}\quad {F.}}} \right)} - 50}\end{matrix}}{2}$

[0051] PLANT:

[0052] Plant Height (to tassel tip): 229.0 cm (SD=6.1)

[0053] Ear Height (to base of top ear): 95.3 cm (4.50)

[0054] Length of Top Ear Internode: 15.9 cm (1.04)

[0055] Average number of Tillers: 0 (0)

[0056] Average Number of Ears per Stalk: 1.0 (0)

[0057] Anthocyanin of Brace Roots: Absent

[0058] LEAF:

[0059] Width of Ear Node Leaf: 9.8 cm (0.83)

[0060] Length of Ear Node Leaf: 65.7 cm (2.4)

[0061] Number of leaves above top ear: 5 (0.60)

[0062] Leaf Angle (from 2nd Leaf above ear at anthesis to Stalk aboveleat): 30.9° (6.6)

[0063] Leaf Color: Medium Green-Munsell Code 5 GY 4/6

[0064] Leaf Sheath Pubescence (Rate on scale from 1=none to 9=like peachfuzz): 1

[0065] Marginal Waves (Rate on scale from 1=none to 9=many): 2

[0066] Longitudinal Creases (Rate on scale from 1=none to 9=many): 3

[0067] TASSEL:

[0068] Number of Lateral Branches: 8 (1.8)

[0069] Branch Angle from Central Spike: 41.4° (5.10)

[0070] Tassel Length (from top leaf collar to tassel top): 40.1 cm (4.6)

[0071] Pollen Shed (Rate on scale from 0=male sterile to 9=heavy shed):7

[0072] Anther Color: Green-Yellow-Munsell Code 2.5 GY 8/6

[0073] Glume Color: Medium Green-Munsell Code 5 GY 5/6

[0074] Bar Glumes: Absent

[0075] EAR:(Unhusked Data)

[0076] Silk Color (3 days after emergence): Light Green-Munsell Code 2.5GY 8/4

[0077] Fresh Husk Color (25 days after 50% silking): Light Green-MunsellCode 2.5 GY 7/8

[0078] Dry Husk Color (65 days after 50% silking): Buff-Munsell Code 7.5YR 7/4

[0079] Position of Ear: Upright

[0080] Husk Tightness (Rate on scale from 1=very loose to 9=very tight):4

[0081] Husk Extension: Short (ears exposed)

[0082] EAR: (Husked Ear Data)

[0083] Ear Length: 16.6 cm (1.4)

[0084] Ear Diameter at mid-point: 43.1 mm (3.6)

[0085] Ear Weight: 107.0 gm (10.4)

[0086] Number of Kernel Rows: 14 (0.80)

[0087] Kernel Rows: Distinct

[0088] Row Alignment: Straight

[0089] Shank Length: 13.0 cm (2.2)

[0090] Ear Taper: Average

[0091] KERNEL: (Dried)

[0092] Kernel Length: 10.5 mm (0.6)

[0093] Kernel Width: 9.5 mm (0.6)

[0094] Kernel Thickness: 6.2 mm (0.6)

[0095] Round Kernels (Shape Grade): 44.1% (2.81)

[0096] Aleurone Color Pattern: Homozygous

[0097] Aleurone Color: White-Munsell Code 2.5Y 8/2

[0098] Hard Endosperm Color: Yellow-Munsell Code 2.5Y 7/8

[0099] Endosperm Type: Normal Starch

[0100] Weight per 100 kernels: 32.2gm (0.29)

[0101] COB:

[0102] Cob Diameter at Mid-Point: 30.0 mm (2.0)

[0103] Cob Color: White-Munsell code 2.5 Y 8/2

[0104] AGRONOMIC TRAITS:

[0105] Stay Green (at 65 days after anthesis) 7 (Rate on scale from1=worst to 9=excellent)

[0106] 0% Dropped Ears (at 65 days after anthesis)

[0107] 0% Pre-anthesis Brittle Snapping

[0108] 0% Pre-anthesis Root Lodging

[0109] 0% Post-anthesis Root Lodging (at 65 days after anthesis)

[0110] The invention provides methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plant,wherein the first or second corn plant is an inbred corn plant from thevariety LH351. Further, both first and second parent corn plants may befrom the inbred variety LH351. Therefore, any methods using the inbredcorn variety LH351 are part of this invention: selfing, backcrosses,hybrid breeding, and crosses to populations. Any plants produced usinginbred corn variety LH351 as a parent are within the scope of thisinvention. Advantageously, the inbred corn variety is used in crosseswith other corn varieties to produce first generation (F) corn hybridseed and plants with superior characteristics.

[0111] Greater test weight, better standability, and higher yields aresome of the advantages LH351 crosses have over LH287 hybrids. Hybridmaturities are one day later than LH287 crosses. A good pollinator,LH351 flowers 2 days later than LH287.

FURTHER EMBODIMENTS OF THE INVENTION

[0112] The invention provides methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plant,wherein either the first or second parent corn plant is an inbred cornplant of the variety LH351. Further, both first and second parent cornplants may be from the inbred corn variety LH351. Still further, theinvention provides methods for producing an inbred corn variety LH351derived corn plant by crossing inbred corn variety LH351 with a secondcorn plant and growing the progeny seed, and repeating the crossing andgrowing steps with the inbred corn variety LH351-derived plant from 0 to7 times. Thus, any such methods using the inbred corn variety LH351 arepart of this invention: selfing, backcrosses, hybrid production, crossesto populations, and the like. All plants produced using inbred cornvariety LH351 as a parent are within the scope of this invention,including plants derived from inbred corn variety LH351. Advantageously,the inbred corn variety is used in crosses with other, different, corninbreds to produce first generation (F₁) corn hybrid seeds and plantswith superior characteristics.

[0113] It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

[0114] As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which corn plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,kernels, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk and the like.

[0115] Duncan, et al., Planta 165:322-332 (1985) reflects that 97% ofthe plants cultured that produced callus were capable of plantregeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, et al., Plant Cell Reports 7:262265 (1988), reportsseveral media additions that enhance regenerability of callus of twoinbred varieties. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao et al., Maize Genetics CooperationNewsletter, 60:64-65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347(1987) indicates somatic embryogenesis from the tissue cultures of cornleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

[0116] Tissue culture of corn is described in European PatentApplication, publication 160,390, incorporated herein by reference. Corntissue culture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 367-372,(1982)) and in Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea Mays Genotypes,” 165Planta 322:332 (1985). Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce corn plantshaving the physiological and morphological characteristics of inbredcorn variety LH1351.

[0117] The utility of inbred corn variety LH351 also extends to crosseswith other species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Croix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with LH351 may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

[0118] With the advent of molecular biological techniques that haveallowed the isolation and characterization of genes that encode specificprotein products, scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred variety.

[0119] Plant transformation involves the construction of an expressionvector which will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed corn plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the corn plant(s).

[0120] Expression Vectors for Corn Transformation

[0121] Marker Genes—Expression vectors include at least one geneticmarker, operably linked to a regulatory element (a promoter, forexample) that allows transformed cells containing the marker to beeither recovered by negative selection, i.e., inhibiting growth of cellsthat do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or a herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

[0122] One commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptll) gene, isolated fromtransposon Tn5, which when placed under the control of plant regulatorysignals confers resistance to kanamycin. Fraley et al., Proc. Natl.Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used selectablemarker gene is the hygromycin phosphotransferase gene which confersresistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol.Biol, 5:299 (1985).

[0123] Additional selectable marker genes of bacterial origin thatconfer resistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

[0124] Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

[0125] Another class of marker genes for plant transformation requirescreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS,3-galactosidase, luciferase and chloramphenicol, acetyltransferase).Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teed et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247:449 (1990).

[0126] Recently, in vivo methods for visualizing GUS activity that donot require destruction of plant tissue have been made available.Molecular Probes publication 2908, Imagene Green™, p. 1-4 (1993) andNaleway et al., J. Cell Biol. 115:151 a (1991). However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

[0127] More recently, a gene encoding Green Fluorescent Protein (GFP)has been utilized as a marker for gene expression in prokaryotic andeukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP andmutants of GFP may be used as screenable markers.

[0128] Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

[0129] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

[0130] Inducible Promoters

[0131] An inducible promoter is operably linked to a gene for expressionin corn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

[0132] Any inducible promoter can be used in the instant invention. SeeWard et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Melt et al., PNAS 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

[0133] Constitutive Promoters

[0134] A constitutive promoter is operably linked to a gene forexpression in corn or the constitutive promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn.

[0135] Many different constitutive promoters can be utilized in theinstant invention. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses such as the 35S promoterfrom CaMV (Odell et al., Nature 313:810-812 (1985) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., PlantJournal 2 (3): 291-300 (1992)).

[0136] The ALS promoter, Xbal/Ncol fragment 5′ to the Brassica napusALS3 structural gene (or a nucleotide sequence similarity to saidXbal/Ncol fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

[0137] Tissue-Specific or Tissue-Preferred Promoters

[0138] A tissue-specific promoter is operably linked to a gene forexpression in corn. Optionally, the tissue-specific promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in corn. Plants transformedwith a gene of interest operably linked to a tissue-specific promoterproduce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

[0139] Any tissue-specific or tissue-preferred promoter can be utilizedin the instant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

[0140] Signal Sequences for Targeting Proteins to SubcellularCompartments

[0141] Transport of protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondroin or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine, during protein synthesis andprocessing, where the encoded protein is ultimately compartmentalized.

[0142] The presence of a signal sequence directs a polypeptide to eitheran intracellular organelle or subcellular compartment or for secretionto the apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis; Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

[0143] Foreign Protein Genes and Agronomic Genes

[0144] With transgenic plants according to the present invention, aforeign protein can be produced in commercial quantities. Thus,techniques for the selection and propagation of transformed plants,which are well understood in the art, yield a plurality of transgenicplants which are harvested in a conventional manner, and a foreignprotein then can be extracted from a tissue of interest or from totalbiomass. Protein extraction from plant biomass can be accomplished byknown methods which are discussed, for example, by Heney and Orr, Anal.Biochem. 114:92-6 (1981).

[0145] According to a preferred embodiment, the transgenic plantprovided for commercial production of foreign protein is corn. Inanother preferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

[0146] Likewise, by means of the present invention, agronomic genes canbe expressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

[0147] 1. Genes That Confer Resistance to Pests or Disease and ThatEncode:

[0148] A. Plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant inbred variety can betransformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistanceto Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. Tomato encodes aprotein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2gene for resistance to Pseudomonas syringae).

[0149] B. A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt b-endotoxin gene. Moreover, DNA molecules encoding b-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

[0150] C. A lectin. See, for example, the disclose by Van Damme et al.,Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

[0151] D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

[0152] E. An enzyme inhibitor, for example, a protease or proteinaseinhibitor or an amylase inhibitor. See, for example, Abe et al., J.Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993)(nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I),Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotidesequence of Streptomyces nitrosporeus a-amylase inhibitor).

[0153] F. An insect-specific hormone or pheromone such as an ecdysteroidand juvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

[0154] G. An insect-specific peptide or neuropeptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expressioncloning yields DNA coding for insect diuretic hormone receptor), andPratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (anallostatin is identified in Diploptera puntata). See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

[0155] H. An insect-specific venom produced in nature by a snake, awasp, etc. For example, see Pang et al., Gene 116:165 (1992), fordisclosure of heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide.

[0156] I. An enzyme responsible for a hyper accumulation of amonterpene, a sesquiterpene, a steroid, hydroxamic acid, aphenylpropanoid derivative or another non-protein molecule withinsecticidal activity.

[0157] J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Bio/. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

[0158] K. A molecule that stimulates signal transduction. For example,see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994),of nucleotide sequences for mung bean calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

[0159] L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

[0160] M. A membrane permease, a channel former or a channel blocker.For example, see the disclosure of Jaynes et al., Plant Sci 89:43(1993), of heterologous expression of a cecropin-R, lytic peptide analogto render transgenic tobacco plants resistant to Pseudomonassolanacearum.

[0161] N. A viral-invasive protein or a complex toxin derived therefrom.For example, the accumulation of viral coat proteins in transformedplant cells imparts resistance to viral infection and/or diseasedevelopment effected by the virus from which the coat protein gene isderived, as well as by related viruses. See Beachy et al., Ann. rev.Phytopathol. 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

[0162] O. An insect-specific antibody or an immunotoxin derivedtherefrom. Thus, an antibody targeted to a critical metabolic functionin the insect gut would inactivate an affected enzyme, killing theinsect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

[0163] P. A virus-specific antibody. See, for example, Tavladoraki etal., Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

[0164] Q. A developmental-arrestive protein produced in nature by apathogen or a parasite. Thus, fungal endo a-1, 4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-a-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10:1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

[0165] R. A development-arrestive protein produced in nature by a plant.For example, Logemann et al., Bio/technology 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

[0166] 2. Genes That Confer Resistance to a Herbicide, For Example:

[0167] A. herbicide that inhibits the growing point or meristem, such asan imidazalinone or a sulfonylurea. Exemplary genes in this categorycode for mutant ALS and AHAS enzyme as described, for example, by Lee etal., EMBO J. 7:1241 (1988), and Miki et al, Theor. Appl. Genet. 80:449(1990), respectively.

[0168] B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyltransferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acct-S1, Accl-S2 and Acct-S3 genes described byMarshall et al., Theor. App/. Genet. 83:435 (1992).

[0169] C. A herbicide that inhibits photosynthesis, such as a triazine(psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla etal., Plant Cell 3:169 (1991), describe the transformation ofChlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker; and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285:173 (1992).

[0170] 3. Genes That Confer or Contribute to a Value-Added Trait, Suchas:

[0171] A. Modified fatty acid metabolism, for example, by transforming aplant with an antisense gene of stearyl-ACP desaturase to increasestearic acid content of the plant. See Knultzon et al., Proc. NatL Acad.Sci. U.S.A. 89:2624 (1992).

[0172] B. Decreased Phytate Content

[0173] 1) Introduction of a phytase-encoding gene would enhancebreakdown of phytate, adding more free phosphate to the transformedplant. For example, see Van Hartingsveldt et al., Gene 127:87 (1993),for a disclosure of the nucleotide sequence of an Aspergillus nigerphytase gene.

[0174] 2) A gene could be introduced that reduced phytate content. Inmaize, this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35:383 (1990).

[0175] C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis a-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley a-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

[0176] Methods for Corn Transformation

[0177] Numerous methods for plant transformation have been developed,including biological and physical, plant transformation protocols. See,for example, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

[0178] A. Agrobacterium-mediated Transformation

[0179] One method for introducing an expression vector into plants isbased on the natural transformation system of Agrobacterium. See, forexample, Horsch et al, Science 227:1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems andmethods for Agrobacterium-mediated gene transfer are provided by Gruberet al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7,1997.

[0180] B. Direct Gene Transfer

[0181] Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

[0182] A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 pm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. TechnoL 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

[0183] Another method for physical delivery of DNA to plants issonication of target cells. Zhang et al., Bio/Technology 9:996 (1991).Alternatively, liposome or spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-ornithine have also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982).

[0184] Electroporation of protoplasts and whole cells and tissues havealso been described. Donn et al., In Abstracts of Vllth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

[0185] Following transformation of corn target tissues, expression ofthe above-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

[0186] The foregoing methods for transformation would typically be usedfor producing a transgenic inbred variety. The transgenic inbred varietycould then be crossed, with another (non-transformed or transformed)inbred variety, in order to produce a new transgenic inbred variety.Alternatively, a genetic trait which has been engineered into aparticular corn line using the foregoing transformation techniques couldbe moved into another line using traditional backcrossing techniquesthat are well known in the plant breeding arts. For example, abackcrossing approach could be used to move an engineered trait from apublic, non-elite inbred variety into an elite inbred variety, or froman inbred variety containing a foreign gene in its genome into an inbredvariety or varieties which do not contain that gene. As used herein,“crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

[0187] When the term inbred corn plant is used in the context of thepresent invention, this also includes any single gene conversions ofthat inbred. The term single gene converted plant as used herein refersto those corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the act that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original inbredof interest (recurrent parent) is crossed to a second inbred(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cornplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

[0188] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original inbred. To accomplish this, a single gene of the recurrentinbred is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0189] Many single gene traits have been identified that are notregularly selected for in the development of a new inbred but that canbe improved by backcrossing techniques. Single gene traits may or maynot be transgenic, examples of these traits include but are not limitedto, male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

INDUSTRIAL APPLICABILITY

[0190] Corn is used as human food, livestock feed, and as raw materialin industry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry milling are grits, meal and flour.The corn wet-milling industry can provide corn starch, corn syrups, anddextrose for food use. Corn oil is recovered from corn germ, which is aby-product of both dry- and wet-milling industries.

[0191] Corn, including both grain and non-grain portions of the plant,is also used extensively as livestock feed, primarily for beef cattle,dairy cattle, hogs and poultry.

[0192] Industrial uses of corn include production of ethanol, cornstarch in the wetmilling industry and corn flour in the dry-millingindustry. The industrial applications of corn starch and flour are basedon functional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The corn starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds and other miningapplications.

[0193] Plant parts other than the grain of corn are also used inindustry, for example: stalks and husks are made into paper andwallboard and cobs are used for fuel and to make charcoal.

[0194] The seed of inbred corn line LH351, the plant produced from theinbred seed, the hybrid corn plant produced from the crossing of theinbred, hybrid seed, and various parts of the hybrid corn plant andtransgenic versions of the foregoing, can be utilized for human food,livestock feed, and as a raw material in industry.

Hybrid Comparisons

[0195] In the table that follows, the traits and characteristics ofinbred corn line LH351 are given in hybrid combination. The datacollected on inbred corn line LH351 is presented for the keycharacteristics and traits. The table presents yield test informationabout LH351. LH351 was tested in several hybrid combinations at numerouslocations, with two or three replications per location. Informationabout these hybrids, as compared to several check hybrids, is presented.

[0196] The first pedigree listed in the comparison group is the hybridcontaining LH351. Information for the pedigree includes:

[0197] 1. Mean yield of the hybrid across all locations.

[0198] 2. A mean for the percentage moisture (% M) for the hybrid acrossall locations.

[0199] 3. A mean of the yield divided by the percentage moisture (Y/M)for the hybrid across all locations.

[0200] 4. A mean of the percentage of plants with stalk lodging (%Stalk) across all locations.

[0201] 5. A mean of the percentage of plants with root lodging (% Root)across all locations.

[0202] 6. A mean of the percentage of plants with dropped ears (% Drop).

[0203] 7. A mean of the plant height (Plant Hgt) in centimeters.

[0204] 8. A mean of the ear height (Ear Hgt) in centimeters

[0205] 9. The number of locations indicates the locations where thesehybrids were tested together.

[0206] The series of hybrids listed under the hybrid containing LH351are considered check hybrids. The check hybrids are compared to hybridscontaining the inbred LH351.

[0207] The (+) or (−) sign in front of each number in each of thecolumns indicates how the mean values across plots of the hybridcontaining inbred LH351 compare to the check crosses. A (+) or (−) signin front of the number indicates that the mean of the hybrid containinginbred LH351 was greater or lesser, respectively, than the mean of thecheck hybrid. For example, a+4 in yield signifies that the hybridcontaining inbred LH351 produced 4 bushels more corn than the checkhybrid. If the value of the stalks has a (−) in front of the number 2,for example, then the hybrid containing the inbred LH351 had 2% lessstalk lodging than the check hybrid.

LH351 Hybrids versus Check Hybrids

[0208] Plant Ear Mean % % % Height Height Test Pedigree Yield % M Y/MStalk Root Drop (cm) (cm) Weight LH 195 × 204 20.7 9.85 5 1 0 110 4456.7 LH351  LH95 × +25 +0.35 +1.08 +1 −1 0 +1 +4 +0.7 LH287 LH310 × +5+0.50 −0.01 −2 −1 0 −4 0 +1.6 LH287 LH195 × +18 +1.13 +0.34 +2 −1 0 +4+3 +0.5 LH185 LH244 × 207 20.1 10.34 5 1 0 118 48 56.1 LH351 LH244 × +4+0.54 −0.06 0 −1 0 +3 +3 +0.5 LH287 LH244 × +9 +0.65 +0.12 +1 0 0 +2 +2−0.2 LH185 LH246 × 212 21.9 9.66 8 1 0 112 44 56.1 LH351 LH246 × +16+0.85 +0.37 +3 0 0 0 0 +0.7 LH287 LH246 × +20 +1.08 +0.47 +3 0 0 +3 +2+0.2 LH185  HC33 × 207 18.9 10.93 4 1 0 116 44 57.0 LH351  HC33 × +7−0.33 +0.54 −2 −1 0 +8 0 −1.8 LH283  HC33 × +3 −0.09 +0.12 0 0 0 +10 +4+0.5 LH287

Deposit Information

[0209] A representative deposit of 2500 seeds of the inbred corn varietydesignated LH351 has been made with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. on (______). Thosedeposited seeds have been assigned ATCC Accession No. ______. Thedeposit was made in accordance with the terms and provisions of theBudapest Treaty relating to deposit of microorganisms and was made for aterm of at least thirty (30) years and at least five (05) years afterthe most recent request for the furnishing of a sample of the deposit isreceived by the depository, or for the effective term of the patent,whichever is longer, and will be replaced if it becomes non-viableduring that period.

[0210] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant inbred and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

What is claimed is:
 1. A seed of the corn variety LH351, wherein asample of the seed of the corn variety LH351 was deposited under ATCCAccession No. ______.
 2. A population of seed of the corn variety LH351,wherein a sample of the seed of the corn variety LH351 was depositedunder ATCC Accession No. ______.
 3. The population of seed of claim 2,further defined as an essentially homogeneous population of seed.
 4. Thepopulation of seed of claim 2, further defined as essentially free fromhybrid seed.
 5. A corn plant produced by growing the seed of claim
 1. 6.A plant part of the corn plant of claim
 5. 7. The plant part of claim 6,further defined as pollen, an ovule or a cell.
 8. A tissue culturecomprising the cell of claim
 7. 9. An essentially homogeneous populationof corn plants produced by growing the seed of the corn variety LH351,wherein a sample of the seed of the corn variety LH351 was depositedunder ATCC Accession No. ______.
 10. A corn plant capable of expressingall the physiological and morphological characteristics of the cornvariety LH351, wherein a sample of the seed of the corn variety LH351was deposited under ATCC Accession No. ______.
 11. The corn plant ofclaim 10, further comprising a nuclear or cytoplasmic gene conferringmale sterility.
 12. A tissue culture of regenerable cells of a plant ofcorn variety LH351, wherein the tissue is capable of regenerating plantscapable of expressing all the physiological and morphologicalcharacteristics of the corn variety LH351, wherein a sample of the seedof the corn variety LH351 was deposited under ATCC Accession No. ______.13. The tissue culture of claim 12, wherein the regenerable cellscomprise cells derived from embryos, immature embryos, meristematiccells, immature tassels, microspores, pollen, leaves, anthers, roots,root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.
 14. Thetissue culture of claim 13, wherein the regenerable cells compriseprotoplasts or callus cells.
 15. A corn plant regenerated from thetissue culture of claim 12, wherein the corn plant is capable ofexpressing all of the physiological and morphological characteristics ofthe corn variety designated LH351, wherein a sample of the seed of thecorn variety LH351 was deposited under ATCC Accession No. ______.
 16. Aprocess of producing corn seed, comprising crossing a first parent cornplant with a second parent corn plant, wherein one or both of the firstor the second parent corn plant is a plant of the corn variety LH351,wherein a sample of the seed of the corn variety LH351 was depositedunder ATCC Accession No. ______, wherein seed is allowed to form. 17.The process of claim 16, further defined as a process of producinghybrid corn seed, comprising crossing a first inbred corn plant with asecond, distinct inbred corn plant, wherein the first or second inbredcorn plant is a plant of the corn variety LH351, wherein a sample of theseed of the corn variety LH351 was deposited under ATCC Accession No.______.
 18. The process of claim 17, wherein crossing comprises thesteps of: (a) planting the seeds of first and second inbred corn plants;(b) cultivating the seeds of said first and second inbred corn plantsinto plants that bear flowers; (c) preventing self pollination of atleast one of the first or second inbred corn plant; (d) allowingcross-pollination to occur between the first and second inbred cornplants; and (e) harvesting seeds on at least one of the first or secondinbred corn plants, said seeds resulting from said cross-pollination.19. Hybrid corn seed produced by the process of claim
 18. 20. A hybridcorn plant produced by growing a seed produced by the process of claim18.
 21. The hybrid corn plant of claim 20, wherein the plant is a firstgeneration (F₁) hybrid corn plant.
 22. The corn plant of claim 5,further defined as having a genome comprising a single locus conversion.23. The corn plant of claim 22, wherein the single locus was stablyinserted into a corn genome by transformation.
 24. The corn plant ofclaim 22, wherein the locus is selected from the group consisting of adominant allele and a recessive allele.
 25. The corn plant of claim 22,wherein the locus confers a trait selected from the group consisting ofherbicide tolerance; insect resistance; resistance to bacterial, fungal,nematode or viral disease; yield enhancement; waxy starch; improvednutritional quality; enhanced yield stability; male sterility andrestoration of male fertility.
 26. A method of producing an inbred cornplant derived from the corn variety LH351, the method comprising thesteps of: (a) preparing a progeny plant derived from corn variety LH351by crossing a plant of the corn variety LH351 with a second corn plant,wherein a sample of the seed of the corn variety LH351 was depositedunder ATCC Accession No. ______; (b) crossing the progeny plant withitself or a second plant to produce a seed of a progeny plant of asubsequent generation; (c) growing a progeny plant of a subsequentgeneration from said seed and crossing the progeny plant of a subsequentgeneration with itself or a second plant; and (d) repeating steps (b)and (c) for an additional 3-10 generations to produce an inbred cornplant derived from the corn variety LH351.